FY2015 has seen significant progress toward accomplishing all of the Specific Aims; some of this progress is detailed here. For Aim 1, the first project focuses on the early development of MS lesions. Previously, we studied two critical aspects of lesion development: the small veins around which white matter lesions form, and the spatiotemporal dynamics of vascular permeability as manifested in gadolinium-enhanced MRI. To understand whether the presence of a central vein may help distinguish MS lesions from their mimickers an idea that remains controversial and to which we only partially subscribe we developed a rapid imaging approach for clinical 3T MRI called FLAIR*. Studies to assess the utility of FLAIR* for diagnosis and characterization of MS lesions are currently underway in our lab and in several other labs worldwide. Preliminary results indicate that the FLAIR* technique is able to significantly improve diagnostic confidence in a variety of settings, and an international workshop is being convened in November 2015 to address this question. With respect to vascular permeability, we have established that there are two spatiotemporal patterns in MS lesions: a centrifugal pattern, in which serum contents leak from the center of the lesion and then proceed outward, over the course of minutes to hours, to fill the entire lesion; and a centripetal pattern, in which serum contents first appear on the periphery of the lesion and then proceed inward. These findings have important implications for understanding lesion development and its association with blood-brain-barrier permeability. In further work, we described how these permeability patterns help to determine the fashion in which acute MS lesions evolve into their chronic counterparts. Specifically, we have found that very early events, perhaps occurring within the first month after lesion formation, appear to determine the efficacy of tissue repair, possibly including remyelination. This finding paves the way for development of a specific clinical trial paradigm for testing new or repurposed agents that might facilitate this aspect of the repair process. Additionally under Aim 1, we have completed and published work on the evolution of inflammatory demyelinating lesions in the brains of marmoset monkeys with experimental autoimmune encephalomyelitis (EAE). We previously demonstrated that the blood-brain barrier becomes locally permeable up to four weeks prior to the onset of demyelination, and we showed that this permeability is associated with a perivascular lymphocytic and mononuclear infiltrate with parenchymal activation of microglia and astrocytes. Ongoing experiments are designed to dissect the cellular and radiological correlates of neuroprotection and lesion repair in marmoset EAE in a fashion that will have direct implications for our human studies. Finally, we completed recruitment of asymptomatic first-degree relatives of MS patients as part of the first stage of the nationwide Genes and Environment in Multiple Sclerosis (GEMS) study, a collaboration with colleagues at the Brigham & Womens Hospital of Harvard University (NCT01353547 and NCT01617395). At NIH, we characterized individuals possibly at relatively high and low risk for development of clinical MS. Preliminary results, which we hope to present publicly within the next year, show subtly but potentially meaningful differences between these two groups. For Aim 2, work in the past year has continued to focus on development of methodology for radiological-pathological correlation studies, particularly in the marmoset EAE model. We implemented high-resolution imaging of formalin-fixed brains using a variety of MRI approaches and developed a system to use those images to guide the histopathological analysis. This is accomplished by generating 3D-printed brain-cutting boxes that allow precise sectioning of the brain, such that small lesions observed on MRI (either in vivo or postmortem) can be localized and studied. We have demonstrated the value of this system for analyzing areas of neocortical demyelination and leptomeningeal inflammation. We have further shown its ability to analyze tiny abnormal disease foci in the marmoset model, which we are in the process of characterizing relative to their cellular components and for the presence or absence of heavy metals. Additional work has focused on development of a clinical trial paradigm for early-phase efficacy testing of new drugs to protect and repair brain tissue undergoing inflammatory demyelination. In this work, we have shown that the results of conventionally but carefully acquired MRI scans, analyzed using sophisticated statistical models, can be used to infer whether such a drug failed to achieve its desired effect. This is important because there is no current method to test such drugs in trials containing fewer than dozens or hundreds of individuals that last a year or more. Our approach holds the promise of greater efficiency and sensitivity, as it can be accomplished in 6 months or less with 20 study participants or potentially even fewer. In FY15, we began initial testing of one new agent, developed by our extramural collaborators, and we plan to start efficacy testing using our new trial design in the upcoming year.